In vitro macrophage activation: A technique for screening anti-inflammatory, immunomodulatory and anticancer activity of phytomolecules.

Macrophage activation plays a significant role in homeostasis of organisms. Various internal and external stress factors may affect their function, leading to adverse effects on the body. 'In vitro macrophage activation techniques provide us with a window to understand the mechanisms of inflammation and response of macrophages to the modulating interventions. Apart from infectious diseases, inflammation is also the major culprit in pathogenesis of many noncommunicable diseases such as arthritis, obesity, metabolic syndrome, diabetes, cancer, cardiovascular disease etc. In vitro macrophage activation allows us to study the role of polarized macrophages in the process of pathogenesis. This emerging technique leads to newer diagnostics, understanding pathophysiological mechanism/s, drug development and management of chronic inflammatory diseases. We, at MRC-KHS, use this technique for screening of medicinal plant-derived phytomolecules for their anti-inflammatory, immunomodulatory and anticancer activities. This review briefly outlines the different experimental models of in vitro macrophage activation and their applications for understanding the pathophysiological mechanisms of underlying chronic inflammation and screening of therapeutic activity of plant-based phytomolecules.

Beta-thalassaemia carrier detection by ELISA: a simple screening strategy for developing countries.

The frequency of beta-thalassaemia in India ranges from 3.5% to 15% in the general population and of the 100,000 children born with thalassaemia major in the world, 10,000 are in India alone. Affected children do not die immediately, but treatment by regular transfusion is costly and leads to iron overload and death. Therefore, health services in lower-economic countries can sustain patients only if the numbers can be limited. Detecting carrier couples by simple blood test can prevent thalassaemia and at-risk couples can be identified and informed of their genetic risk before having children. A prevention programme including population screening, counselling, and prenatal diagnosis will markedly reduce the birth prevalence of affected individuals. Hemoglobin A2 (HbA2) measurement in human hemolysates has great significance, since its level can indicate beta-thalassaemia carrier status in otherwise healthy individuals. We have developed a rapid, simple, and inexpensive enzyme linked immunosorbent assay (ELISA) for the quantitation of HbA2, which can be used in carrier screening programmes in developing countries like India. In a limited trial for beta-thalassaemia carrier screening, the results obtained with ELISAs were compared with those obtained with the microcolumn chromatography method (r = 0.89).

Identification and purification of an aspartic proteinase from human semen.

To purify and evaluate the molecular changes associated with an aspartic protease (Cathepsin D) in human semen from infertile subjects. Cathepsin D was purified from normo-, oligo- and azoospermic semen, by a procedure involving detergent solubilisation, affinity chromatography and gel filtration chromatography. The enzyme from normo-, oligo- and azoospermic samples was purified 86, 60 and 44 fold respectively. The purified enzyme appeared as a single band on SDS as well as on native PAGE irrespective of the pathological conditions. The molecular weight of Cathepsin D from oligospermic and normospermic samples was 40 kDa while that of azoospermic sample was found to be 43 kDa. The enzyme was inhibited by pepstatin while other proteinase inhibitors and metal ions did not have any effect. Purified Cathepsin D from azoospermic sample differs from normospermia and oligospermia.

Lipid peroxidation and antioxidant enzymes in male infertility.

BACKGROUND AND AIM: Mammalian spermatozoa are rich in polyunsaturated fatty acids and are very susceptible to attack by reactive oxygen species (ROS) and membrane lipid peroxide ion. Normally a balance is maintained between the amount of ROS produced and that scavenged. Cellular damage arises when this equilibrium is disturbed. A shift in the levels of ROS towards pro-oxidants in semen and vaginal secretions can induce an oxidative stress on spermatozoa. The aim was to study lipid peroxidation and antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase (SOD) and to correlate the same, with the 'water test', in male infertility. SETTINGS: Experimental study. SUBJECTS AND METHODS: Ejaculates from a total of 83 infertile and fertile healthy individuals were obtained. Lipid peroxidation and antioxidant enzyme levels were studied and correlated with water test. RESULTS: The results indicate that (i) the antioxidant enzyme catalase showed no significant changes in the various pathological samples, (ii) antioxidant enzymes SOD and glutathione peroxidase correlate positively with asthenozoospermic samples and (iii) the degree of lipid peroxidation also correlates positively with the poorly swollen sperm tails. The increase in SOD and glutathione peroxidase values, in the pathological cases represents an attempt made to overcome the reactive oxygen species. CONCLUSION: Water test could be used as a preliminary marker test for sperm tail damage by reactive oxygen species, since it correlates very well with lipid peroxidation and antioxidant enzymes.

Correlation between creatine kinase activity, lipid-peroxidation and water test in male infertility.

New approaches need to be pursued towards the assessment of sperm quality using biochemical markers. In order to help develop a good biochemical marker to assess sperm-membrane integrity, the enzyme creatine kinase (CK) was studied in semen of normal, oligospermic and azoospermic samples and correlated with sperm concentration, lipid-peroxidation (LP) and water test. Presence of isoforms of creatine kinase (CK-MB) was also seen. An inverse correlation was observed between CK activity and sperm concentration (p<0.001). Water test was seen to be inversely correlated with CK activity (p<0. 001). Lipid peroxidation showed positive correlation with CK activity (p<0.001). A significant correlation between loss of sperm function meditated by induction of peroxidative damage to sperm plasma membrane is indicated. Enzymes like CK can serve as good biochemical marker along with lipid peroxidation to confirm loss of sperm membrane integrity. The water test can be used as a preliminary screening test for sperm membrane integrity.

Seminal profiles of lysosomal enzymes in normal and infertile men.

Human seminal plasma is known to possess considerable proteolytic activity, much of which is associated with lysosomes. The activities of lysosomal hydrolases like alkaline proteinase, cathepsin-D, aryl-sulfatase and N-acetyl-beta-D-glucosaminidase in seminal plasma from randomly chosen infertile and vasectomised men have been compared. These enzymes have been implicated in the coagulation and liquefaction processes. The role of fructose and proteins in these processes has also been studied. The results indicate that cathepsin-D and aryl-sulfatase activity in infertile men were significantly lower than normo-spermic subjects. N-acetyl-beta-D-glucosaminidase was lowest in azoospermia suggesting that it could be used as a biochemical marker for azoospermia. Conversely, alkaline proteinase showed increased levels in all the infertile cases.

Correlation between serum immunoreactive inhibin, gonadotrophins and spermatogenesis.

Serum inhibin, LH and FSH were measured in infertile married men. A negative correlation between FSH/LH ratio and serum inhibin levels was observed. Comparison of serum inhibin and FSH levels with testicular histology indicated an association between the formation of spermatids and the elevation of inhibin levels. A plot of serum inhibin levels versus testicular inhibin levels (ng/100 mg protein) showed an excellent correlation.

Levels of immunoreactive inhibin-like material in urine during the menstrual cycle.

Using a specific and sensitive radioimmunoassay, the authors determined levels of inhibin-like material in the urine of eight healthy women with normal menstrual cycle length of 28 +/- 4 days. The results revealed a cyclic variation in urinary immunoreactive inhibin levels during the menstrual cycles, with a sharp rise in levels three to four days prior to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) peaks. These levels of immunoreactive inhibin may thus serve as a parameter to detect impending LH surge.

PIP: Using a specific and sensitive radioimmunoassay, the authors determined the levels of inhibinlike material in the urine of 8 healthy women with normal menstrual cycle length of 28 +or- 4 days. The results revealed a cyclic variation in urinary immunoreactive inhibin levels during the menstrual cycles, with a sharp rise in levels 3-4 days prior to luteinizing hormone (LH) and follicle-stimulating hormone peaks. These levels of immunoreactive inhibin may thus serve as a parameter to detect impending LH surge.

Inhibin interaction with LHRH receptors at the pituitary level.

In order to investigate whether inhibin could modulate the action of luteinizing hormone releasing hormone (LHRH), the in vitro effect of inhibin of LHRH bindings to the pituitaries from intact adult male rats was studied. The inhibin preparations suppressed the binding of labeled LHRH to pituitary receptors in a dose-related manner. In vivo administration of inhibin decreased the pituitary LHRH receptor concentration, with an apparent increase in the affinity of these receptors. A dose-related decrease was observed in serum follicle stimulating hormone (FSH) levels in the same group whereas the serum luteinizing hormone (LH) levels remained unaltered. There may be a direct action of inhibin at the pituitary level to suppress FSH levels specifically.

Biological studies with low and high molecular weight inhibin preparations.

The effects of both high and low molecular weight inhibin preparations on testicular and pituitary receptors were studied. Both these preparations were able to inhibit the binding of labelled hFSH to testicular receptor in a dose related manner, but were unable to affect the binding of labelled hCG to its receptor, suggesting that the observed inhibition of FSH binding was specific to inhibin. In addition, the binding of LHRH to pituitary receptors was affected by inhibin preparations. Interestingly, the antiserum raised against high molecular weight inhibin was able to neutralize, totally, the biological effect of high molecular weight inhibin, and only, partially, the biological effect of low molecular weight inhibin.

Occurrence of bio-immuno active inhibin in rat spermatids.

Levels of inhibin in the spermatocyte and spermatid enriched fractions of rat testicular extract were estimated using a specific and sensitive radioimmunoassay. The fraction enriched in spermatids had the highest amounts of inhibin activity. The results of the study indicate that the spermatids may be likely source of inhibin.